The complement system is a complex group of proteins in body fluids that, working together with antibodies or other factors, play an important role as mediators of immune, allergic, immunochemical and immunopathological reactions. Activation of the complement system can result in a wide range of reactions such as lysis of various kinds of cells, bacteria and protozoa, inactivation of viruses, and the direct mediation of inflammatory processes. Through the hormone-like activity of several of its components, the complement system can recruit and enlist the participation of other humoral and cellular effector systems. These in turn can induce directed migration of leukocytes, trigger histamine release from mast cells, and stimulate the release of lysosomal constituents from phagocytes.
The complement system consists of at least twenty distinct plasma proteins capable of interacting with each other, with antibodies, and with cell membranes. Many of these proteins when activated combine with some of the other proteins to form enzymes to cleave and activate still other proteins in the system. The sequential activation of these proteins follows two main pathways, the classical pathway and the alternative pathway. Both pathways use a common terminal trunk which leads to cell lysis or virus inactivation.
The classical pathway can be activated by antigen-antibody complexes, aggregated immunoglobulins and non-immunological substances such as DNA and trypsin-like enzymes. The classical pathway of activation involves, successively, four components denominated C1, C4, C2 and C3. These components can be grouped into two functional units: C1 or recognition unit; and C4, C2, and C3 or activation unit. Five additional components denominated C5, C6, C7, C8, and C9 define the membrane attack unit forming the terminal truck common to both pathways.
In the classical pathway, C1 is activated such as by attachment to an immunoglobulin and through a series of reactions produces an activated C1s from a constituent of C1. A bar over the term for a complement factor denotes an active enzyme. Activated C1s cleaves portions of both of components C4 and C2. Parts of the C4 and C2 components then combine to form the activated complex C4b,2a having a molecular weight of about 280,000. C4b,2a is a proteolytic enzyme which continues ongoing complement action. Earlier components are no longer required after it has been formed. C4b,2a cleaves and thereby activates the next component of the sequence, C3, to produce C3b which attaches to cell membranes adjacent to the C4b,2a. The C3b then combines with the C4b,2a to form the last activated complex in the classical pathway C4b,2a,3b. This enzyme cleaves C5, a component of the membrane attack unit.
The alternative pathway, also known as the properdin pathway, comprises at least six components. Five of these components truly belong to the alternative pathway, factors B, D, properdin (P), and two inhibitors, H and I. The sixth component, C3, can also be found in the classical pathway. Component C3b is sometimes also known as factor A. The alternative pathway can be activated by immunological substances such as IgA and nonimmunological substances such as certain complex polysacharides, trypsin-like enzymes and cobra venom factor. Even in the absence of any antibody or immunoglobulin, the alternative pathway can destroy microorganisms.
Activation of the alternative pathway proceeds in a different manner than the classical pathway. An initial requirement is the presence of C3b which appears to be continuously generated in small amounts in the body. C3b production is thought to be due to water induced cleavage of a thioester bond in C3 forming an activated C3* which reacts with the factors B and D to generate an enzyme to cleave C3 into C3a and C3b. C3b can be further produced by a positive feedback mechanism in which factor D and Bb (a component of factor B) combine with C3b to form the activated complex C3b,Bb that acts as an enzyme in an amplification loop to cleave more C3 to form additional C3b. Factors I and H act as regulator proteins by cleaving C3b to render it inactive. Other regulator proteins include C1 inhibitor and C4 binding protein.
C3b,Bb enzyme molecules are rendered more efficient by properdin (P) which binds to the complex and stabilizes it by slowing the spontaneous disociation of factor Bb. Both C3b,Bb and C3b,P,Bb cleave additional C3 molecules to form modified poly-C3b enzymes, C3b.sub.n,Bb and C3b.sub.n,P,Bb, wherein "n" is greater than 1. Any of these molecules can also cleave C5 into C5a and C5b and initate the membrane attack unit of the same common terminal trunk. The C5b then combines with C6 and C7 to form an active trimolecular complex, C5b,6,7. The C5b,6,7 then combines with C8 and a plurality of C9's to form a further, active complex, which on the surface of a cell causes cytolysis.
Study and measurement of the activation of a complement pathway can provide an indication of many possible biological disorders. The two complement pathways have been implicated in the pathogenesis or symptomatology of a broad spectrum of human diseases and pathologic conditions. In the case of the classical pathway, these include immune complex diseases of several types, autoimmune diseases, in particular systemic lupus erythematosus, and infectious diseases. The alternative pathway has been found to be involved in infections with gram negative bacteria, viruses, parasites, and fungi, gram negative septicemia, and various dermatologic, renal, and hematologic diseases. Alternative pathway activation has also been associated with trauma, burns and adult respiratory distress syndrome (ARDS), as well as contact with dialysis membranes such as during hemodialysis and cardiac bypass surgery. In vitro studies have indicated that a number of gram negative bacteria and bacterial products, virus infected cells, viruses, protozoa, fungi, burns, damaged and injured cells, and other substances of biomedical importance have the ability to activate the alternative pathway in human serum.
Present methods to assess and quantitify complement pathway function and activation are indirect, limited in number, and generally only available in laboratories engaged in research on the complement pathways. They measure not the dynamic activity of a pathway, but rather a static end state or the capability of the pathway. One such crude screening test for an intact complement sequence in human serum is hemolytic assay. Hemolytic assay is used to calculate the CH50 level, the point at which 50 percent of the antibody-coated erythrocytes (EA) in a test sample are lysed by a particular dilution of serum containing the complement components. This method is rather insensitive because it relies on a secondary event, lysis, and does not measure pathway activation directly, but rather residual functional activity of the complement system. Hemolytic assay also cannot measure the activity of any particular component produced in the activation sequence, only the total activation. Activation and, by implication, functional ability of the entire complement sequence are necessary to result in lysis.
The presence of individual complement components in blood sera can be measured by the use of antibodies prepared against the appropriate complement component. However, this only gives an indication of the amount of complement component present in sera and not the amount of activation of a pathway. Previous attempts to measure the presence of only an activated component require complicated separation techniques. See Cooper, "Laboratory Evaluation of Complement Activation" in Immunoassays: Clinical Laboratory Techniques for the 1980's at pp. 393-410, R. M. Nakamura, W. R. Dito, and E. S. Tucker III, editors, Alan R. Liss Inc., New York, N.Y. (1980)
Previous techniques to detect and assess activation of the alternative complement pathway have generally been of two types. The first type involves demonstration of reduced functional activity of components of the alternative pathway such as C3 and factor B, in human sera after blocking the classical pathway. See for example, Perrin et al., J. Exp. Med., 143:1027-1041 (1976); Ferrone et al., Proc. Natl. Acad. Sci. USA., 70: 3665-3668 (1973). A variant of this method is to assess the deposition of components of the alternative complement pathway in diseased tissue. See Verroust et al., J. Clin. Invest., 53:77-84 (1974). Such methods suffer from the following limitations: , (a) the alternative complement pathway activation is not directly measured, rather only the secondary consequences of activation, (b) multiple purified complement components must be prepared in the testing laboratory which must also possess facilities that verify functional activity, and, (c) the method does not permit quantification.
The second type of method used to measure activation of the alternative pathway detects the deposition of components of the alternative pathway such as C3 or factors B and H on the surface of the activator particle. See Schreiber et al., Proc. Natl. Acad. Sci. USA., 75:3948-3952 (1978). A variant of this procedure is to measure the specific ratios of these components such as the factor H to C3 ratio. See Pangburn et al., J. Immunol, 124:977-982 (1980).
While activation is directly assessed in the above procedure and can be quantified, there are certain limitations. These limitations include (a) the requirement to purify and radiolabel multiple complement components in the testing laboratory and the associated requirement for facilities to verify functional integrity, (b) the involved techniques and interpretation of results are complex and require intimate familiarity with the system and, (c) the approach cannot be used to detect and quantify preexisting activation, and thus cannot be used with sera or plasma samples from patients.
Other attempts have been directed to the detection and measurement of inactivated products such as C1 inactivator bound to subcomponents of C1. See Harpel et al. Clin. Res., 30: 563A (1982) and Hack et al., J. Immun., 127: 1459 (1981). However, these systems are not directed to activated complexes which continue complement activity. Rather they are relative "dead end" products whose presence is not necessary indicative of the amount of complement pathway activation. The detection of these products was performed because they are known to be relatively stable and therefore available for assay. However, such products can remain as residuals in the blood and can be formed when there is no further activation of a complement system.
Further background information on the operation and measurement of the complement system can be found in Cooper, "The Complement System" in Basic and Clinical Immunology, pp. 124-135, Stites et al. editors, Lange Medical Publications, Los Altos, Calif. (1982); H. Rapp and T. Borsos, Molecular Basis of Complement Action, pp. 81-83, Appleton-Century Crofts, New York, N.Y. (1970); Muller-Eberhard, et al., Adv. Immunol., 29:1-53 (1980); Pangburn et al., J. Immunol., 124:977-982 (1980); Schreiber et al., Clin. Immunol. and Immunopathol., 15:384-396 (1980); Platts-Mills et al., J. Immunol., 113:348-357 (1974); Lesavre et al., J. Immunol., 123:529-534 (1979); Polhill et al., J. Immunol., 121:363-370 (1978); Fearon et al., J. Immunol., 115:1357-1361 (1975); Day et al., Scand. J. Immunol., 5:715-720 (1976); Chapitis et al., J. Exp. Med., 143:241-257 (1976).
It would be desirable to provide a method and system which avoids the difficulties of the prior art procedures and provides for effective detection and quantification of activation of the complement system. Such detection of activation would be directed not to a whole sequence or a regulator protein, but to an activated complex which is indicative of the dynamic activity of a pathway. It would also be desirable if such a system and method were relatively easy to use and highly specific to the complement complex being assayed. The system and method should also be sensitive and able to detect relatively small amounts of complement activation. The present invention meets these desires.